Methods and compositions with trans-clomiphene

ABSTRACT

The present invention relates to the use of compositions comprising trans-clomiphene for treating men with hypogonadism. The invention is also directed to methods for treating males with hypogonadism. The present invention is also directed to methods of decreasing cholesterol levels.

This application is a continuation-in-part of International ApplicationNo. PCT/US02/021524, filed Jul. 9, 2002, which claims the benefit, under35 U.S.C. § 119(e), of U.S. Provisional Patent Application No.60/304,313, filed on Jul. 9, 2001.

FIELD OF THE INVENTION

The present invention relates to the compositions and methods forincreasing testosterone levels. More specifically, the present inventionrelates to a composition comprising clomiphene enriched fortrans-clomiphene. The present invention also relates to the use of acomposition comprising clomiphene enriched for trans-clomiphene reagentsfor increasing testosterone levels.

BACKGROUND

Testosterone is the primary male androgen, playing a vital role inoverall male health. Testosterone is essential to the development andmaintenance of specific reproductive tissues (testes, prostate,epididymis, seminal vesicle, and penis) and male secondary sexcharacteristics. It plays a key role in libido and erectile function andis necessary for the initiation and maintenance of spermatogenesis.Testosterone also has important functions not related to reproductivetissues. For example, it positively affects body composition byincreasing nitrogen retention, which supports lean body mass, musclesize and strength. It also acts on bone to stimulate bone formation.

Testosterone secretion is the end product of a series of hormonalprocesses. Gonadotropin-releasing hormone (GnRH), which is secreted inthe hypothalamus, controls the pulsatile secretion of luteinizinghormone (LH) and follicle stimulating hormone (FSH), which are secretedby the anterior pituitary. LH, in turn, regulates the production andsecretion of testosterone in the Leydig cells of the testes, while FSHassists in inducing spermatogenesis.

Testosterone is most often measured as “total testosterone.” Thismeasurement includes testosterone that is bound to sex hormone-bindingglobulin (SHBG) (˜44%) and is therefore not bioavailable andtestosterone which either is free (˜2%) or loosely bound to otherproteins (non-SHBG-bound) (˜54%).

Results from a WHO study indicate that testosterone is normally secretedin a circadian rhythm, with higher levels in the morning and nadirlevels occurring around 8 to 10 p.m. See FIG. 1. This variation intestosterone secretion throughout the day becomes much less pronouncedin older men (mean age equals 71 years). The importance of this rhythmis not known at this time.

Samples were obtained from both young and elderly patients every 10minutes for 24 hours via an indwelling cannula. According to Tenover(1987) the mean 24 hr total serum testosterone levels in healthy youngmen (age range 22 yrs.-35 yrs. mean 27.3 yrs) was 4.9±0.3 (±SEM) mg/ml(17.0 nmol/L) while older men (age range 65 yrs-84 yrs. mean 70.7 yrs.)had a significantly lower mean 24 hrs. total serum testosterone level of4.1±0.4 mg/ml. (P<0.5; 14.2 nmol/L).

Total serum testosterone levels obtained from single random samples werealso significantly lower in older men (4.0±0.2 mg/ml [13.9 nmol/L]) ascompared to 4.8±0.2 mg/ml [16.6 nmol/L] in healthy young men.

Testosterone deficiency can result from underlying disease or geneticdisorders and is also frequently a complication of aging. For example,primary hypogonadism results from primary testicular failure. In thissituation, testosterone levels are low and levels of pituitarygonadotropins (LH and FSH) are elevated. Secondary hypogonadism is dueto inadequate secretion of the pituitary gonadotropins. In addition to alow testosterone level, LH and FSH levels are low or low-normal. Some ofthe sequelae of adult testosterone deficiency include a wide variety ofsymptoms including: loss of libido, erectile dysfunction, oligospermiaor azoospermia, absence or regression of secondary sexualcharacteristics, progressive decrease in muscle mass, fatigue, depressedmood and increased risk of osteoporosis.

Several forms of testosterone therapy exists in the United States today.Recently, transdermal preparations have gained favor in the market.However, a scrotal testosterone patch results in supraphysiologic levelsof 5α-dihydrotestosterone (DHT) due to the high concentration of5α-reductase in scrotal skin. It is not known whether these elevated DHTlevels have any long-term health consequences. Nonscrotal systems areconsidered more convenient and most patients achieve average serumconcentrations within the normal range and have normal levels of DHT.Oral testosterone therapy is not recommended because doses required forreplacement therapy are associated with significant risk ofhepatotoxicity.

SUMMARY

The present invention is directed to compositions useful for increasingtestosterone levels in male mammals and for ameliorating or preventingthe sequelae of low testosterone levels. In one of its aspects theinvention is directed to compositions having active ingredientscomprising 0% to 29% weight/weight of (cis, -Z-, trans-clomiphene)(hereinafter “cis-clomiphene”) and 100% to 71% w/w (trans-, E-,cis-clomiphene) (hereinafter “trans-clomiphene”) or pharmaceuticallyacceptable salts thereof. Among the preferred compositions of thepresent invention which contain both cis-clomiphene and trans-clomipheneinvention are compositions wherein the ratio of trans-clomiphene andcis-clomiphene is greater than 71/29. A more preferred compositionaccording to the present invention comprises about 100% w/w of activeingredients of trans-clomiphene or a pharmaceutically acceptable saltthereof. All compositions of the present invention may further comprisesuitable pharmaceutical excipients diluents, carriers, and the like.Analogs of the cis-clomiphene and trans-clomiphene are also contemplatedfor use in all aspects of the present invention.

The present invention is also directed to methods for increasing serumtestosterone levels in hypogonadal male mammals (and for ameliorating orpreventing the sequelae of low testosterone levels), the methodcomprising administering to a subject male an effective amount of acomposition according to the present invention, the compositions havingactive ingredients comprising 0% to 29% weight/weight of cis-clomipheneand 100% to 71% w/w trans-clomiphene including any of theirpharmaceutically acceptable salts thereof. Among the preferred methodsare those in which the administered compositions contain both isomerswherein the ratio of trans-clomiphene to cis-clomiphene is greater than71/29. A more preferred method comprises administering to the male acomposition comprising about 100% w/w of trans-clomiphene.

The present invention is also directed to compositions useful fordecreasing cholesterol levels in mammals, and more specifcally in malemammals, and for ameliorating or preventing the sequelae of highcholesterol levels. More preferably, the present invention is directedto using compositions having active ingredients comprising 0% to 29%weight/weight of cis-clomiphene and 100% to 71% w/w of trans-clomipheneor pharmaceutically acceptable salts thereof in treating highcholesterol levels in mammals, and more specifically in male mammals.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graphic representative of the normal secretory total serumtestosterone profiles in healthy men (young and old).

FIG. 2 shows the chemical structure of clomiphene citrate.

FIG. 3 is a graphic demonstration of the time course of serumtestosterone levels with Clomid, Enclomid and Zuclomid.

FIG. 4 is a graphic demonstration of the time course of cholesterollevels in baboon males treated with Clomid, Enclomid and Zuclomid.

DETAILED DESCRIPTION

The present invention provides methods and compositions useful forincreasing testosterone levels in male mammals and for ameliorating orpreventing the sequelae of low testosterone levels including but notlimited to those described above.

Clomiphene (FIG. 2) is an antiestrogen related to tamoxifen that blocksthe normal estrogen feedback on the hypothalamus and subsequent negativefeedback on the pituitary. This leads to increases in luteinizinghormone (LH) and follicle stimulating hormone (FSH). In men, theseincreased levels of gonadotropins stimulate the Leydig cells of thetestes and result in the production of higher testosterone levels.Clomiphene citrate has the following structure:

Ernst et al., J. Pharmaceut. Sci. 65:148 (1976), have shown thatclomiphene is a mixture of two geometric isomers which they refer to ascis, -Z-, clomiphene (cis-clomiphene or zuclomiphene) and trans-,E-,clomiphene, (trans-clomiphene or enclomiphene). According to Ernst, etal. trans-clomiphene HCl has a melting point of 149° C.-150.5° C., whilecis-clomiphene HCl has a melting point of 156.5° C.-158° C.

Ernst et al. have also noted that (the trans-isomer) is antiestrogenic(AE) while the cis-isomer is the more potent and more estrogenic formand has also been reported to have anti-estrogenic activity. The authorsattribute the effect of the drug on ovulatory activity to both formsstating that the mixture is more effective than trans-clomiphene alone.The trans-isomer aids ovulation at the level of the hypothalamus. Theestrogenic isomer cis-clomiphene contributes to enchanced ovulationelsewhere in the physiologic pathway leading to ovulation. The isomersare also reported to have different in vivo half-life. Furthermore thecis form has been reported to leave residual blood levels for in excessof one month following a single dose.

Vandekerckhove, et al. (Cochrane Database Syst Rev 2000;(2):CD000151(2000)) noted that ten studies involving 738 men have suggested thatanti-estrogens appear to have a beneficial effect on endocrinaloutcomes, i.e. testosterone, but there is not enough evidence toevaluate fertility effects. Nevertheless should clomipheneadministration enhance testosterone levels then one could easilyconclude that the drug should positively impact the side effects oftestosterone deprivation as long as the testes still retain the abilityto respond to gonadotropin stimulation.

Clomiphene is currently approved as a mixture of both cis- andtrans-isomers, the cis-isomer being present as about 30% to 50% (MerckManual) for fertility enhancement in the anovulatory patient. Clomipheneimproves ovulation by initiating a series of endocrine eventsculminating in a preovulatory gonadotropin surge and subsequentfollicular rupture. The drug is recommended to be administered for 5days at a dose of up to 100 mg daily. Clomiphene has also beenassociated with numerous side effects including: blurred vision,abdominal discomfort, gynecomastia, testicular tumors, vasomotorflushes, nausea, and headaches. Furthermore, other studies suggest thatclomiphene possesses both genotoxic and tumor enhancement effects. Thenet outcome of these observations is that clomiphene in its currentformat, having between 30% and 50% of the cis isomer, would beunacceptable for chronic therapy in men for the treatment oftestosterone deficiency.

Clomiphene has also been used for therapeutic intervention in men withlow testosterone levels. Tenover et al., J. Clin. Endocrinol. Metab.64:1103, (1987) and Tenover et al., J. Clin. Endocrinol. Metab. 64:1118(1987) found increased in FSH, LH in both young and old men aftertreatment with clomiphene. They also found increases in free and totaltestosterone in men with young men showing significant increases.

Studies were also conducted to determine whether or not clomiphene couldbe used to improve fertility in men by improving semen quality. Homonnaiet al. Fertil. and Steril 50:801 (1988) saw increases in spermconcentration and count but others have not. (See e.g., Sokel, et al.,Fertil. and Steril. 49:865 (1988); Check, et al, Int. J. Fertil. 34:120(1989); Purvis, et al., Int. J. Androl 21:109 (1989); and Breznik, Arch.Androl. 21:109 (1993).) One group saw a deterioration in the percentageof normal sperm with long-term treatment. Shamis, et al., Arch. Androl21:109 (1991). A WHO study showed no changes in semen quality orfertility after 6 months of treatment. (Anonymous Androl. 15:299(1992).) A meta-analysis seems to confirm that testosterone levels go upin men with poor quality sperm but not fertility. (Vanderkerckhove, etal., 2000). Studies have also suggested that long term treatment withclomiphene does not seem to have a drastic deleterious effect on health,although it did show that treatment resulted in poorer sperm qualityafter 4 months. Studies have kept men on clomiphene for as long as 18months and at levels of 25 mg per day or 100 mg every other day.

In 1991, Guay et al (Urology 38:377 (1991)) suggested that clomiphenecould treat sexual dysfunction in men. Their hypothesis seems to be thatsexual function follows testosterone levels. This was supported by earlystudies showing positive influence of androgens and sexual function,Davidson, et al., J. Clin. Endocrinol. Metab. 48:955 (1979), and studiesthat rated sleep-related erections as a strong response to T,Cunningham, et al., J. Clin. Endocrinol. Metab. 70:792 (1990). However,in 1995, Guay et al. (Gray, et al., J. Clin. Endocrinol. Metab. 80:3546(1995)) published a study in which they saw increase in LH, FSH, andtestosterone after 2 months of clomiphene but no effects on erectiledysfunction. There might be some advantage for young men and specificgroups of older men, but it seems that just raising the testosteronelevel is not enough. Effects of testosterone on sleep-related erectionsmay have been taken too seriously (Herskowitz, et al., J. Psychosomat.Res. 42:541 (1997)).

According to the present invention, a composition comprising of oneisomer preferably trans-clomiphene or a predefined blend of the isomersof clomiphene as described below differing from the normally producedmixture are used to enhance testosterone levels while reducing the sideeffects of the drug. Thus, the present invention provides an oraltherapy for increasing testosterone levels, which lacks or hasdiminished side effects connected with the existing clomipheneformulations.

In one embodiment of the present invention, a patient who has a need ordesire to increase their serum testosterone levels are administered oneor more dosages of an effective amount of composition comprisingtrans-clomiphene at a dosage between one mg to about 200 mg (althoughthe determination of optimal dosages is with the level of ordinary skillin the art). Cis-clomiphene may also be present in the composition solong as the ratio of trans-clomiphene to cis-clomiphene is greater than71/29. Analogs of the trans- and cis-isomers of clomiphene such as thosedescribed in Ernst, et al. supra are also useful in the practice of thepresent invention.

Dosages are preferably (but not necessarily) administered as part of adosage regimen designed to give rise to serum testosterone levels thatmimic or correspond to the normal secretary total serum testosteroneprofile described in FIG. 1. For example, according to FIG. 1 a dosageof the preferred composition may be administered in a pharmaceuticalformulation that would give rise to peak serum testosterone levels ataround 8 a.m. Such pharmaceutical formulations may be in the form ofsustained release formulations prepared as described for example in U.S.Pat. No. 6,221,399, Japanese patent 4-312522, Meshali et al, Int. J.Phar. 89:177-181 (1993), Kharenko et al, Intern. Symp. Control Rel.Bioact. Mater. 22:232-233 (1995), WO 95/35093, Dangprasit et al., Drug.Devel. and Incl. Pharm. 21 (20):2323-2337 (1995); U.S. Pat. Nos.6,143,353, 6,190,591, 6,096,338, 6,129,933, 6,126,969, 6,248,363 andother sustained release formulations well known in the art.

In another embodiment of the present invention, one or more dosages ofan effective amount of composition comprising trans-clomiphene at adosage between one mg to about 200 mg are administered to a patient whohas a need to decrease their serum cholesterol levels. The patient maybe a male or a female. Cis-clomiphene may also be present in thecomposition as long as the ratio of trans-clomiphene to cis-clomipheneis greater than 71/29. Analogs of the trans- and cis-isomers ofclomiphene such as those described in Ernst, el at. supra are alsouseful in the practice of the present invention.

Suitable pharmaceutical compositions or unit dosage form may be in theform of solids, such as tablets or filled capsules or liquids such assolutions suspensions, emulsions, elixirs or capsules filled with thesame, all for oral use. The compositions may also be in the form ofsterile injectable solutions or emulsions for parenteral (includingsubcutaneous) use. Such pharmaceutical compositions and unit dosageforms thereof may comprise ingredients in conventional proportions.

Compositions according to the present invention may also be administeredby the intravenous, subcutaneous, buccal, transmucusal, intrathecal,intradermal, intracisternal or other routes of administration. Afteradministration of the composition serum testosterone levels may bemeasured as described above and dosages may be altered to achieve asufficient increase in the serum testosterone levels to achieve thedesired physiological results associated with normal testosteronedescribed above.

All of the references discussed herein are incorporated by reference intheir entirety.

The following Examples are meant to be illustrative of the invention andis not intended to limit the scope of the invention as set out in theappended claims.

EXAMPLE 1 Effects of Clomids on Serum Testosterone and Cholesterol inMale Baboons

Adult, male, Baboons were given 1.5 mg/kg of Clomid, Enclomid(trans-Clomid) or Zuclomid (cis-Clomid) for 12 consecutive days. Thesamples analyzed were sera taken on the day of first treatment beforebeing given test article (day 0), after 12 days of treatment (day 12)and 7 days after the last treatment (end or wash-out).

1. Effects on Body Weight and Serum LH, FSH, PRL and Testosterone

There were significant increases in total serum testosterone in thegroup receiving Enclomid. See Table 1. There were no differences amonggroups in the baseline period or at day 0. There were also nodifferences among the three groups 7 days after treatment (the washoutperiod). However, Enclomid produced higher levels of testosteronecompared to Clomid and Zuclomid on day 6 (p=0.03 and p=0.00002respectively) and compared to Zuclomid on day 12 (p=0.047). Zuclomidclearly did not raise total serum testosterone to any extent. Comparedto the animals receiving Enclomid, the animals receiving Clomidexhibited more variable total testosterone levels on day 6 and later asjudged by their coefficients of variations. When we looked at the timecourse of the effects (FIG. 3), we determined that only Enclomidsignificantly and statistically raised total serum testosterone on days6 and 12 compared with either baseline or day 0 values. Moreover,cessation of Enclomid treatment, resulted in a significant drop in thelevel of total serum testosterone between day 12 and day 18 (washout).This indicates that Enclomid is readily cleared from the circulationconsistent with the metabolic clearance seen for Enclomid in humans.Enclomid was clearly better and more consistent than Clomid itself andZuclomid was ineffective.

TABLE 1 Serum Testosterone Levels (ng/dl) baseline 0 day 6 days 12 dayswash-out Group ID Dec. 3, 2001 Dec. 7, 2001 Dec. 13, 2001 Dec. 20, 2001Dec. 26, 2001 CLO 7500 79.01 76.15 940.97 891.5 150.9 9012 97.55 305.24585.92 555.6 316.3 9097 158.06 102.94 151.12 318.9 143.6 mean 111.5161.4 559.3 588.7 203.6 SD 41.3 125.2 395.6 287.7 97.7 ENCLO 7223 64.5774.96 1223.8 633.6 307.2 8021 166.86 133.59 1128.2 1466 399.2 8369170.45 106.47 1081.1 1166 271 mean 134.0 105.0 1144.4 1088.5 325.8 SD60.1 29.3 72.7 421.6 66.1 ZUCLO 7438 124.84 210.4 137.51 314.5 359.78292 104.66 67.37 169.98 406.1 860.5 10098  282.29 904.82 227.95 353.0274.1 mean 170.6 394.2 178.5 357.9 498.1 SD 97.3 448.0 45.8 46.0 316.8ANOVA p = 0.61 p = 0.43 p = 0.007 p = 0.57  p = 0.256 K-W p = 0.56 p =0.84 p = 0.051 p = 0.079 p = 0.252

There were no changes in serum LH or FSH. The ratio of total serumtestosterone to LH followed the same pattern as total serumtestosterone, suggesting a lack of dependence (data not shown). Therewas also no change in body weight during the 12 day study. There was adecrease in serum prolactin (PRL) during the study in the groupreceiving Enclomid, suggesting an effect of antiestrogen that has beendescribed in part (Ben-Jonathan and Hnasko, 2001) and expected on thebasis of the fact that as men age, testosterone declines and Prolactinincrease (Feldman et al., 2002).

2. Effects on Cholesterol Levels

Treatment with Enclomid tended to decrease serum cholesterol andZuclomid tended to increase the same parameter. Preliminary analysisindicated that the changes in cholesterol levels were not statisticallysignificant and that the changes were within the normal range. Due tothe observed trend for the two isomers to demonstrate opposite effectson cholesterol levels over a short period of time, further analysis wasconducted.

Detailed analysis indicated that Enclomid resulted in an 8% decrease inserum cholesterol levels. Conversely, treatment with Zuclomid resultedin a 22% increase in serum cholesterol levels. Treatment with Clomidresulted in a slight increase in serum cholesterol levels. The oppositeeffect of Enclomid and Zuclomid on serum cholesterol levels is notunexpected given that the isomers have, alternatively, estrogen agonistor antagonist activity. These results indicate that Enclomid may be usedfor treating patients with high cholesterol levels. These results alsoindicate that Enclomid may be more benign than Zuclomid with respect toserum cholesterol if used chronically for increasing testosteronelevels.

3. Effects on Clinical Chemistry Parameters

The mean values for each parameter did not differ among the three groupsfor any test parameter at the beginning of the study as determined byANOVA or by the Kruskal-Wallis test. All groups exhibited normal valuesat each parameter except for (1) serum sodium; a related calculatedparameter, anionic gap, which were low for all nine baboons throughoutthe trial; (2) serum glucose; and (3) BUN which were high on day 0 forthe group which would be treated with Enclomid. On day 12 of treatmentand 7 days after treatment (washout), there were no differences amonggroups for any parameter except anionic gap that showed that the Clomidand Zuclomid groups had lower values than the Enclomid group. The valuesof serum sodium and anionic gap appear to be anomalies associated withthis group of baboons.

There were substantive effects on the red blood cell population withEnclomid and Zuclomid and on hematocrit with Zuclomid. All the compoundslower the mean cell hemoglobin concentration (MCHC) either at day 0 orat the endpoint. With no change in mean cell hemoglobin (MCH) and anincrease in the mean cell volume (MCV), the lowering of MCHC ispredictable. Although testosterone might be expected to raisehematocrit, only Zuclomid treatment, which did not increase total serumtestosterone, demonstrated a statistical difference. Clearly, men in aclinical trial that uses Zuclomid should be monitored for thecharacteristics of their red blood cell population. Enclomid would bepredicted to have less of an effect.

There appears to be a clear effect of 12-day Enclomid treatment onplatelets although the values found stayed within the normal range. Onething to consider here is the sexual dimorphism in platelet countsbetween male and female baboons (279 for males vs. 348 for females).This is likely to be due to hormones. Since the Enclomid groupdemonstrated increased testosterone, the lowering of the platelet countcould be secondary to the change in testosterone in this group.Moreover, treatment with Enclomid pushed the platelet count to itsnormal male level from a day 0 level that was the high end of the normalrange for this group. Enclomid would not necessarily predict adeleterious effect on platelets.

All the Clomids tested had effects on the white blood cell (WBC)population, the most striking was that of Enclomid on raising the countsof lymphocytes and eosinophiles. The effects are not as straightforwardas they would seem to be. There appears to be a strong effect ofEnclonud on lowering the percent of granulocytes in the blood. Theeffects are very strong after the 7-day washout period when the valuesare decreased below the normal range. (This time course could reflectthe relatively long time required to affect change the WBC population.)There is little sexual dimorphism in baboons with respect to the whiteblood cell populations, so the effects are more likely to be due to thecompound itself than changes in testosterone. However, when we look atthe calculated count of granulocytes using the WBC count, we find nodifferences in granulocyte count due to any compound. Concomitantly, itis the lymphocyte story that is the most interesting. Both the count andpercent lymphocytes in the population increase with Enclomid treatment.Whereas the mean values of percent lymphocytes remain in the normalrange, given the trend for an increase in WBC count, the net effect isan increase in lymphocyte count with Enclomid. This eosinophil result isanalogous. There is a clear implication for treating men who have lowlymphocytes, such as men who are HIV-positive. Since Enclomid isunlikely to lower lymphocytes based on this result, a case could be madefor its use in the population of men with AIDS. These individuals areoften treated with agents that are intended to raise testosterone due tothe wasting effects of disease. Low liver and kidney toxicity andfavorable effects on cholesterol and lipids are also highly favoredattributes for any medication intended for use HIV-positive men who arealready compromised by their disease.

The increase in serum glucose with Clomid or Zuclomid was within thenormal range. In the case of Enclomid where the mean serum glucosevalues were high on day 0, there were no increases with treatment. Therewas no evidence that Enclomid would have a deleterious effect on bloodglucose.

No clearly adverse effects on liver function are apparent as judged bythe enzymes AST and ALT. The trend in these values was a decrease withtreatment. An increase in the level of enzymes in the serum wouldindicate liver damage. ALT/SGPT was out of range low at the end of thestudy for the Clomid group although the differences over the treatmentperiod were not statistically significant. The changes with Enclomid andZuclomid were within the normal range. AST is depressed in pregnancy;thus the action of an estrogen agonist such as Zuclomid in lowering themarginal AST level could be rationalized. Alkaline phosphatase (ALP) isalso found in the liver and is elevated various disease states. Thelowering of ALP argues further against hepatic damage. There were nochanges in serum albumin, also a liver product. A strong suppression ofserum albumin over an extended time period could contribute to freeserum steroid hormone levels in humans although a more important role isplayed by sex hormone binding globulin. As a bottom line, none of thecompounds could be linked to liver damage on the basis of the parametersassayed.

Osteoblastic activity and diseases of the bone are accompanied by highserum ALP values. ALP was not elevated following Zuclomid treatment andwas decreased in value following Enclomid treatment. The trends wouldpredict a more benign result for the use of Enclomid compared toZuclomid.

Although BUN and BUN/creatinine were altered during the study in theClomid and Enclomid groups, the lack of a definitive change increatinine argues against renal dysfunction. A loss of glomerularfiltration capacity would result in an increase in BUN. Decreased BUNoccurs in humans due to poor nutrition (not likely in a controlledsetting), or high fluid intake (presumably accompanied by edema). Also,despite an increase in total serum testosterone between day 0 and Day 12with Enclomid, there were no differences between serum creatininevalues, arguing against an increase in muscle mass over this short timeinterval.

Serum sodium levels were lower than reference values for all animalsthroughout the study. Serum carbon dioxide was higher than referencevalues on day 12 for the Clomid and Zuclomid groups. Serum anion gap waslower for all animals throughout the study, paralleling the sodiumresults. Enclomid raised this parameter towards normal values. Theelectrolyte imbalances detected in the test animals throughout alltreatment periods remains elusive but might be part of the same fluidderangement phenomenon suggested by the BUN results.

The foregoing results indicate that Enclomid is more effective thanClomid or Zuclomid at enhancing total serum testosterone. Zuclomid isclearly not effective and that deficiency limits any use of Clomid forhypogonadism, particularly since the Zuclomid component of Clomid wouldpredominate in the circulation over time given its longer half-life.

Enclomid appeared to be relatively benign in all aspects when comparedto Zuclomid and, often, even Clomid. This is particularly true whenconsideration is given to the trend of Enclomid to lower cholesterol,and liver enzymes as opposed to Zuclomid's trend to raise the sameparameters. The surprising trend for Enclomid to raise the lymphocytecount may be useful for men with AIDS if it can be shown the CD4+subpopulation of lymphocytes is not lowered or is enhanced.

EXAMPLE 2 Method for Increasing Testosterone Level in Men UsingTrans-clomiphene and Mixtures of Trans-clomiphene and Cis-clomiphene atRatios Greater Than 71/29

Prior to administration of trans-clomiphene, blood samples are takenfrom subject males and testosterone levels are measured usingmethodologies described for example in Matsumoto, et al. Clin.Endocrinol. Metab. 56; 720 (1983) (incorporated herein by reference).Sex hormone binding globulin (SHBG), both free and bound totestosterone, may also be measured as described for example in Tenoveret al. J. Clin. Endocrinol. Metab. 65:1118 (1987) which describemeasurement of SHBG by both a [³H] dihydrotestosterone saturationanalysis and by radioimmunoassay. Non-SHBG-bound testosterone levels(bioavailable testosterone) are also measured for example according toTenover et al. J. Clin. Endocrinol and Metab. 65:1118 (1987). See alsoSoderguard et al J. Steroid Biochem 16:801 (1982) incorporated herein byreference.

Patients are given daily dosages of 1.5 mg/kg clomiphene, wherein theratio of trans-clomiphene to cis-clomiphene is greater than 71/29.Patients are monitored for testosterone levels such that the dosageamount and dosage frequency may be adjusted to achieve therapeuticlevels of testosterone in the patient.

EXAMPLE 3 Method for Decreasing Cholesterol Levels in Men UsingTrans-clomiphene and Mixtures of Trans-clomiphene and Cis-clomiphene atRatios Greater Than 71/29

Prior to administration of trans-clomiphene, blood samples are takenfrom subject males and cholesterol levels are measured usingmethodologies described for example in Cooper, et al. (Selected methodsfor the small clinical chemistry laboratory. W. R. Faulkner and S.Meites, eds. Am. Assoc. for Clin. Chem., Washington, D.C. Pages165-174), which is incorporated herein by reference.

Patients are given daily dosages of 1.5 mg/kg clomiphene, wherein theratio of trans-clomiphene to cis-clomiphene is greater than 1. Thedosage amount and dosage frequency of clomiphene may be adjusted toachieve therapeutic levels of cholesterol in the patient by monitoringfor cholesterol levels on different days of the treatment.

EXAMPLE 4 Method for Decreasing Cholesterol Levels in Women UsingTrans-clomiphene and Mixtures of Trans-clomiphene and Cis-clomiphene atRatios Greater Than 71/29.

Patients are given daily dosages of 1.5 mg/kg clomiphene, wherein theratio of trans-clomiphene to cis-clomiphene is greater than 71/29. Thedosage amount and dosage frequency of clomiphene may be adjusted toachieve therapeutic levels of cholesterol in the patient by monitoringfor cholesterol levels on different days of the treatment.

1. A method of decreasing cholesterol levels in a human, the methodcomprising administering to a human in need thereof, an effective amountof a composition comprising about 100% w/w of active ingredients oftrans-clomiphene or pharmaceutically acceptable salts or solvatesthereof and optionally one or more pharmaceutically acceptable diluents,adjuvants, carriers or exicipients.
 2. The method of claim 1 where thehuman is a male.
 3. The method of claim 1 where the human is a female.4. The method of claim 1, wherein the composition is administered in adosage of 1-200 mg of trans-clomiphene per day.
 5. The method of claim4, wherein the composition is administered in a dosage of about 50 mg oftrans-clomiphene per day.
 6. The method of claim 4, wherein thecomposition is administered in a dosage of 1.5 mg/kg of trans-clomipheneper day.
 7. The method of claim 1 wherein the composition is provided incapsule form.